Accurate cell division requires the proper assembly and function of microtubule-based structures such as the bipolar spindle needed to segregate chromosomes, and the spindle midzone (or central spindle) required to keep segregated chromosomes apart and help position the site of cell cleavage. These micron-sized structures require both motor and non-motor proteins to move and organize microtubules, as well as to regulate the formation of new filaments. Our long-term goal is to decipher the molecular mechanisms underlying the assembly and function of these essential structures. To achieve this goal we take a multidisciplinary approach that combines biochemical, structural, biophysical, chemical and cell biological methods. We will build on recent publications and our unpublished preliminary data to focus on the following three Aims: (1) Analyze centrosome-independent microtubule formation. In particular, we will examine how augmin, a recently discovered hetero-octameric protein complex, contributes to microtubule formation within the metaphase spindle. We will employ biochemical, electron microscopy-based, and TIRF (total internal reflection fluorescence) microscopy-based approaches to elucidate how augmin nucleates branched arrays of parallel microtubules. (2) Examine mechanisms regulating microtubule organization. Specifically, we will dissect how PRC1, a non-motor protein that selectively crosslinks antiparallel microtubules, and kinesin-4, a microtubule plus-end directed motor protein that can suppress microtubule growth and disassembly, contribute to the assembly of the spindle midzone, a specialized array of overlapping microtubules that is rapidly assembled during anaphase. We will combine structural, TIRF-microscopy-based, cell biological, and real-time confocal microscopy-based approaches to examine how these two proteins, which bind each other to form a stable complex in solution, regulate microtubule dynamics and antiparallel crosslinking in dividing human cells. (3) Analyze the micromechanics of microtubule arrays. In particular, we will measure how much force is generated to slide microtubules relative to each other by ensembles of motor proteins. TIRF- and optical trap-based assays will be used to determine how the magnitude of forces generated by motor proteins that can crosslink and slide two microtubules apart depends on filament orientation and overlap length. Together, our findings should advance our understanding of how conserved nanometer-sized proteins build the micron-sized structures needed for the stable propagation of our genomes through multiple cell divisions. Errors in cell division have been linked to developmental defects and diseases in humans. Our research should shed light on the molecular mechanisms that ensure the fidelity of chromosome segregation and cytokinesis. Blocking cell division with chemotherapeutic agents is a mainstay of cancer treatments. New anti-cancer drugs are being developed that inhibit cell division by targeting the proteins we study. It is possible that our proposed research will help decipher how these drugs work and may also suggest new targets for therapeutic agents.